The neuromuscular junction (NMJ) is a highly specialized synapse whose formation is dependent on MuSK (muscle-specific kinase), a receptor tyrosine kinase that is activated by agrin, a heparan-sulfate proteoglycan derived from motor neurons. MuSK activation results in the redistribution of muscle proteins to the postsynaptic site, including the acetylcholine receptor (AChR), rapsyn, ErbBs and MuSK itself. In addition, agrin-induced MuSK activation leads to selective transcriptional upregulation of synapse-specific genes in subsynaptic nuclei, and to induction of a retrograde signal leading to presynaptic differentiation. In mature NMJs, acetylcholine released from motor neurons activates AChRs and triggers muscle contraction. Both the ectodomain and cytoplasmic domain of MuSK play essential roles in signaling processes leading to formation of the NMJ. However, the molecular/structural mechanisms underlying these signaling processes are not understood. The goals of this proposal are to elucidate the structural mechanisms governing: (i) MuSK activation by agrin;(ii)MuSK co-clustering with the AChR and rapsyn, and (iii)MuSK recruitment of the non-receptor tyrosine kinases Src and Abl. The specific aims of this proposal are: Aim 1. Structural and functional characterization of the MuSK ectodomain Aim 2. Molecular characterization of the modes of interaction between MuSK and the non-receptor tyrosine kinases Src and Abl To achieve these aims, we will employ x-ray crystallography to determine the three-dimensional structures of the MuSK ectodomain and cytoplasmic domain. We will then characterize the functional roles of key residues identified from the structural studies by expressing select MuSK mutants in M/SK-deficient myotubes and assaying for agrin-induced MuSK activation, AChR clustering, and recruitment of Src and Abl to MuSK. Relevance: Formation of the NMJ is a fundamental biological process which is critical for organismal development. By revealing the biochemical mechanisms underlying the formation of this important synapse, we will better understand, at a molecular level, how defects in this process give rise to neuromuscular disorders.